PhosphoProteomics

Our PhosphoProteomics & PhosphoProtein Array platform offers high sensitivity detection and accurate quantitation of protein phosphorylation. Furthermore, customers can choose to target all 3 types of phosphorylation (pSer/pThr/pTyr) by 2D DIGE PhosphoProtein Array, or a specific type by 2D Phospho Western Blot.

Main applications:

  • Kinase/Phosphatase substrate mapping
  • Kinase/Phosphatase activator screening and identification (such as drug induced protein phosphorylation)
  • Kinase/Phosphatase inhibitor screening and identification
  • Signal transduction studies (regulation of protein functions by phosphorylation)

Here are some highlighted features of our PhosphoProteomics platform:

  • Phospho-protein enrichment (optional)
  • Effective separation of phospho-proteins from their unmodified form by high resolution large format 2D gel
  • High sensitivity detection and quantitation of phospho-proteins by fluorescent labeling
  • Accurate quantitation of changes in protein phosphorylation between different samples
  • Identification of phosphorylation sites is available as a follow up experiment (optional)

Based on your project objective, choose one of the following study designs:

A. Identify proteins differentially phosphorylated (all 3 types: Phospho-Ser, Thr & Tyr)

Study design for 2 samples (Control, Test)

  • Gel-1: Control, Phospho-profiling
  • Gel-2: Test, Phospho-profiling
  • Gel-3: Control, Test

Image reports:

  • Gel-1: Total Protein image, PhosphoProtein-image and overlay image of Total Protein/PhosphoProtein
  • Gel-2: Total Protein image, PhosphoProtein-image and overlay image of Total Protein/PhosphoProtein
  • Gel-3: Protein image of each sample and overlay of the 2 samples

Gel 1:

Control: Total Protein

Phosphoproteomics study design A: protein image of Control sample

Control: Phosphoprotein

Phosphoproteomics study design A: phospho-protein image of Control sample

Total Protein / Phosphoprotein

Phosphoproteomics study design A: protein/phospho-protein overlay image of Control sample

Gel 2:

Test: Total Protein

Phosphoproteomics study design A: protein image of Test sample

Test: Phosphoprotein

Phosphoproteomics study design A: phospho-protein image of Test sample

Total Protein / Phosphoprotein

Phosphoproteomics study design A: protein/phospho-protein overlay image of Test sample

Gel 3:

Control: Total Protein

Phosphoproteomics study design A: protein image of Control sample

Test: Total Protein

Phosphoproteomics study design A: protein image of Test sample

Control / Test

Phosphoproteomics study design A: Control/Test overlay image

Quantitation report:

  • Phospho-ratio: Gel-1 & 2 will provide phospho-signal of each phosphorylated spots and phospho-ratio between 2 samples
  • Protein ratio: Gel-3 will provide the protein ratios
  • Final Phospho-ratio: Calculated by adjusting Phospho-ratios with protein ratios
Assigned IDPhospho RatioProtein RatioFinal Phospho Ratio
13.110.923.39
24.230.944.48
34.370.855.11
46.160.926.71
54.551.064.29

B. Identify proteins differentially phosphorylated at Ser or Thr or Tyr by 2D Western Blot

Study design for 2 samples

  • Gel-1: Control, Western Blot with anti-pSer or pThr or pTyr antibody
  • Gel-2: Test, Western Blot with anti-pSer or pThr or pTyr antibody
  • Gel-3: Control, Test

Image reports:

  • Gel-1: Total Protein image, PhosphoProtein WB image and overlay image of Total Protein/PhosphoProtein
  • Gel-2: Total Protein image, PhosphoProtein WB image and overlay image of Total Protein/PhosphoProtein
  • Gel-3: Protein image of each sample and overlay of the 2 samples

Quantitation report:

  • Phospho-ratio: Gel-1 & 2 will provide phospho-signal of phospho-spots and phospho-ratio between 2 samples
  • Protein ratio: Gel-3 will provide the protein ratios
  • Final Phospho-ratio: Calculated by adjusting Phospho-ratios with protein ratios

Examples of 2D Western Blot with Anti-pSer or Anti-Tyr

Example 1. Anti-Phospho-Tyrosine (pTyr) Western Blot

Jurkat cells were treated with serum starvation for 1 hour. Then serum was added to the medium and cultured for 4 hours. Proteins were extracted by 2D lysis buffer following Applied Biomics protocols. First, sample was run on 2D DIGE (pH4-7) and the gel image was scanned using Typhoon 9400 (A). Second, proteins were transferred to the membrane and Western Blot was performed using mouse monoclonal anti-pTyr antibody (Clone PY20) and CF488-labeled goat anti-mouse Ig (Biotium, Hayward, California). The Western Blot image was scanned using Typhoon 9400 (B) .

A. Cell Lysate Total Protein

Phosphoproteomics study design B: Total protein image

B: Anti-pTyr Western Blot

Phosphoproteomics study design B: Anti-Phospho-Tyrosine (pTyr) Western Blot image

C. Total Protein / Anti-pTyr WB

Phosphoproteomics study design B: Total protein and Tyrosin Phosphorylated protein overlay image

Example 2. Anti-Phospho-Serine (pSer) Western Blot

Hela cells were treated with 1 mM sodium orthovanadate for 30 minutes. Proteins were extracted by 2D lysis buffer following Applied Biomics protocols. First, sample was run on 2D DIGE (pH4-7) and the gel image was scanned using Typhoon 9400 (A). Western Blot was performed using mouse monoclonal anti-pSer antibody (Sigma) and CF488-labeled goat anti-mouse Ig (Biotium, Hayward, California). The Western Blot image was scanned using Typhoon 9400 (B).

A. Cell Lysate Total Protein

Phosphoproteomics study design B: Total protein image

B: Anti-pSer Western Blot

Phosphoproteomics study design B: Anti-Phospho-Serine (pSer) Western Blot image

C. Total Protein / Anti-pSer WB

Phosphoproteomics study design B: Total protein and Serine Phosphorylated protein overlay image

Example 3. Anti-Phospho-Serine/Threonine/Tyrosine (pSer/pThr/pTyr) Western Blot

Hela cells were treated with 1 mM sodium orthovanadate for 30 minutes. Proteins were extracted by 2D lysis buffer following Applied Biomics protocols. First, sample was run on 2D DIGE (pH4-7) and the gel image was scanned using Typhoon 9400 (A). Western Blot was performed using mouse monoclonal antibodies against pSer/pTyr/pThr (Sigma) and CF488-labeled goat anti-mouse Ig (Biotium, Hayward, California). The Western Blot image was scanned using Typhoon 9400 (B).

A. Cell Lysate Total Protein

Phosphoproteomics study design B: Total protein image

B: Anti-pSer/pThr/pTyr WB

C. Total Protein / Anti-pSer/pThr/pTyr

Phosphoproteomics study design B: Total protein and Serine/Threonine/Tyrosine Phosphorylated protein overlay image

C. Identify proteins that are phosphorylated

Study design for 1 sample

Gel-1: Cell lysate, Phospho-profiling or Phospho-WB

A. Cell Lysate Total Protein

Phosphoproteomics study design C: total cell lysate protein image

B: PhosphoProtein

Phosphoproteomics study design B: PhosphoProtein image

C. Total Protein / PhosphoProtein

Phosphoproteomics study design B: Total protein and PhosphoProtein overlay image

Study design for 2 samples:

The phospho-signal is the combined effects of both samples. If you want to obtain phospho-signal for each sample, please use Study design A.

Gel-1: Normal, Drug treated, Phospho-profiling or Phospho-WB

Image report (for 2 samples):

Gel-1: Protein image of each sample and overlay of the 2 samples; Phospho-image of 2 samples and overlay image of Protein/Phospho

1. Normal

Phosphoproteomics study design C: total protein image of Normal sample

2. Drug Treated

Phosphoproteomics study design C: total protein image of Drug-treated sample

3. PhosphoProtein Array

Phosphoproteomics study design C: PhosphoProtein image of combined Normal & Drug-treated samples

Normal / Drug Treated

Phosphoproteomics study design C: Normal & Drug-treated total protein overlay image

Normal / PhosphoProtein Array

Phosphoproteomics study design C: Normal sample total protein & PhosphoProtein overlay image

Normal / Treated / Phosphoprotein

Phosphoproteomics study design C: Normal total protein & Drug-treated total protein & PhosphoProtein triple color overlay image

Quantitation report:

  • Protein ratios will be provided on differently expressed protein spots
  • Phopsho-spots will be marked on the protein ratio table

PhosphoProteomics Applications

1. Drug induced phospho-protein change

Identify Phospho-protein markers due to drug treatment: 2D DIGE was performed on 2 samples (1. Control, 2. Drug treated), followed by phospho-profiling. Data showed that Phospho-protein level increased after drug treatment, while un-phospho protein levels remain similar between the 2 samples.

Control: Protein

Drug induced phospho-protein change: protein image of Control sample

Phosphoprotein

Drug induced phospho-protein change: Phosphoprotein image

Protein / Phospho

Drug induced phospho-protein change: protein & PhosphoProtein overlay image of Control sample

Figure 1: Protein image, Phospho-image, and Protein/Phospho overlay image of Control sample.

Drug-treated: Protein

Drug induced phospho-protein change: protein image of Treated sample

Phosphoprotein

Drug induced phospho-protein change: Phosphoprotein image

Protein / Phospho

Drug induced phospho-protein change: protein & PhosphoProtein overlay image of Treated sample

Figure 2: Protein image, Phospho-image, and Protein/Phospho overlay image of Drug-treated sample

Control + Drug-treated

Drug induced phospho-protein change: Control & Drug-treated protein overlay image

Phosphoprotein

Drug induced phospho-protein change: Phosphoprotein image with PhosphoProteins in purple circles

Control/Treated/Phospho

Drug induced phospho-protein change: Control protein & Drug-treated protein & PhosphoProtein triple color overlay image

Figure 3: Protein image, Phospho-image, and Protein/Phospho overlay image of Control/Drug-treated. Spots circled in purple are phospho-spots up-regulated by drug treatment. Spots circled in white are Non-phospho-spots of the same protein which remain un-changed with drug treatment.

2. Time-dependent phospho-protein changes

Identify phospho-protein changes in a time course: 2D DIGE and Phospho-profiling was performed on Embryonic stem (ES) cells differentiated at Days 0, 2, 4, 6, 8 and 10 (D0-D10). Data showed that Phospho-isoforms of Aconitate hydrase spots expressed at different levels during the time course.

Time-dependent phospho-protein change in stem cells: Phosphorylated Aconitate hydrase image during time course

Figure 1: Black / white image of Phosphorylated Aconitate hydrase (spot 1-3) and non-Phosphorylated Aconitate hydrase (spot 4) at each time point.

Time-dependent phospho-protein change in stem cells: comparison of Aconitate hydrase at different time points vs. day 0

Figure 2: Color overlay image of each time point / D0.

Time-dependent phospho-protein change in stem cells: comparison of Aconitate hydrase at different time points vs. prior time points

Figure 3: Color overlay image of each time point / prior time point

Figures 1-3 indicate that Phosphorylated Aconitate hydrase (spot 1-3) changed over the time course, with the biggest change at Day 4 & 6. In contrast, non-Phosphorylated Aconitate hydrase (spot 4) remain at a similar level at different days. Please see Table 1 for protein ratios.

Table 1: Protein ratios of Aconitate hydrase (spot 1-4).

Embryonic Stem Cell (ES)D2 / D0D4 / D2D6 / D4D8 / D6D10 / D8
#1Phospho-Aconitate hydrase-1.02-1.05+3.28-1.81-1.11
#2Phospho-Aconitate hydrase-1.41+1.41+3.54-1.96-1.06
#3Phospho-Aconitate hydrase-1.45+1.91+1.50-1.09-1.03
#4Non-phospho-Aconitate hydrase-1.45+1.91+1.28-1.07-1.03

Sample Info

Please follow these general principles regarding Biohazard material and buffer condition in getting your samples ready.

Protein amount: 500 -700 µg of total protein from each sample. If you order Phospho-Protein Enrichment (Item 450A and 450N), 5000 µg of total protein from each sample is needed. This will be sufficient to cover the entire procedure including Phospho-Protein Array, preparative gel, spot picking and protein ID.

Protein concentration: 5-20 mg/ml is preferred.

Buffer: Please feel free to submit samples in whichever buffer that you would normally use. The standard sample preparation is covered by the DIGE gel cost. Such samples should have protein concentration in the range of 5-20 mg/ml. Additional charge may apply on samples that require extensive amount of the extra work such as protein eluting, concentrating, buffer exchange or serum abundant protein depletion.

Sample type: Please inform our scientists about your sample type. We will send you additional tips for each sample type.

Pricing

Services DescriptionAcademic/Governmental LabsIndustrial/Commercial Labs
CodePriceCodePrice
2D Phospho-Protein Profiling 1401A-PP1$1500401N-PP1$1800
2D DIGE Phospho-Protein Profiling 1401A-PP2$1900401N-PP2$2280
Phospho-Protein Enrichment (from 5mg)450A$700450N$1000

1. Price covers:

  • Experimental design
  • Standard sample preparation and protein concentration determination
  • Fluorescence dye labeling using CyDye
  • IEF and SDS-PAGE
  • Scanning of protein images using Typhoon scanner
  • Phosphoprotein-staining of the same gel
  • Scanning of protein/Phospho images using Typhoon scanner
  • Three hour data analysis: Image analysis using ImageQuant and manual labeling of phosphorylated spots
  • Data Report

Prices are discounted and only apply for customers doing follow up Protein ID service with Applied Biomics.

Price does NOT cover:

  • Sample preparation that requires extensive amount of the extra work such as protein eluting, concentrating, buffer exchange or serum abundant protein depletion
  • Screen shots of 3D view of each spot
  • Preparative gel, spot picking, protein ID: Please note that in most cases, Phospho-gels (similar to Analytical gels) do not contain sufficient amount of protein for protein identification. If you find some interesting spots you would like to move forward to perform Protein ID, we will need to run a Preparative gel. Please view here for differences between Analytical and Preparative gels. Please click here for the cost of Preparative gel, spot picking and protein ID
Services DescriptionAcademic/Governmental LabsIndustrial/Commercial Labs
CodePriceCodePrice
2D DIGE Preparative Gel (1 CyDye labeling)* 1401A$800401N$960
2D DIGE Preparative Gel (2 CyDye labeling)* 1102A2$1200102N2$1500
2D DIGE Preparative Gel (3 CyDye labeling)* 1102A3$1295102N3$1595
Spot Picking per Gel* 2103$250 per 96 spots103$250 per 96 spots
Protein ID by Mass Spectrometry (MALDI-TOF/TOF) 3104A$125 per spot104N$140 per spot
Pathways Analysis After Protein ID 4203Acall for a quote203Ncall for a quote

*Price applies for customers who perform protein identification at Applied Biomics

1. Price covers:

  • Experimental design
  • Sample preparation and protein concentration determination
  • Fluorescence dye labeling using CyDye (1 sample for 1 CyDye labeling; 2 samples for 2 CyDye labeling; 3 samples for 3 CyDye labeling)
  • IEF and SDS-PAGE
  • Gel image scan using Typhoon scanner
  • Spot picking design

2. Price covers:

  • Set up spot picking
  • Pick spots
  • Free storage at -80C for 6 months.

1-3. Prices are discounted and only apply for customers doing 2D DIGE & protein ID service with Applied Biomics. Turnaround time starting from Preparative gel to sending protein ID report is 7-10 business days. We offer high sensitivity protein identification from gel spots or bands using the latest technologies in mass spectrometry. In each run, four sensitivity standards in the amount of 1-10 femtomole are included. Our sensitivity is the range of 1-2 femtomole. Please view the protein ID procedure for detailed steps of this service. Price covers the following:

  • Gel treatment
  • In-gel trypsin digestion
  • Peptide extraction
  • Desalting
  • Spotting
  • MALDI-TOF
  • MALDI-TOF/TOF
  • Database search against NCBI database using MASCOT
  • Data report (example below)

4. Pathway Analysis Service is provided only for customers doing the 2D DIGE and Protein ID services with Applied Biomics. The report will include the major pathway list with the following information:

  • Functional groups
  • Genes in each functional group
  • Enrichment score
  • Statistic p-value and FDR